It has become apparent that a variety of pathophysiological states are associated with a marked increase in the vascular production of reactive oxygen species. Our work over the past decade has shown that a membrane bound NAD(P)H oxidase is a predominant source of these molecules. A major stimulus for activation of this enzyme is angiotensin II, and we have found that production of superoxide by the NAD(P)H oxidase is crucial for the development of hypertension in response to this octapeptide. In the next funding period, we plan to pursue three new directions related to these fundamental findings. In project 1, Dr. Griendling and co-workers will examine in detail how the vascular smooth muscle NAD(P)H oxidase functions to produce free radical oxygen- and H2O2. During the past funding period, Dr. Griendling and her collaborators cloned a new class of NAD(P)H oxidase subunits, termed the nox proteins, and have shown that nox1 is expressed in vascular cells. Using both cultured cells and genetically altered mice, the interactions of nox1 with other NAD(P)H oxidase subunits will be investigated and the importance of nox1 for vascular free radical oxygen production in vivo will be examined. In project 2, Dr. Harrison will study factors that modulate expression of the extracellular superoxide dismutase (ecSOD). Preliminary data indicate that the ecSOD plays a crucial role in modulation of hypertension in response to angiotensin II and that the expression of this enzyme is increased in conditions of vascular oxidative stress. Dr. Harrison will examine factors responsible for the increase in ecSOD in these conditions, and will investigate mechanisms underlying the augmented hypertension that occurs in mice lacking ecSOD. Dr. Taylor, the director of the last project, has previously shown that angiotensin II plays a crucial role in exacerbating atherosclerosis in the setting of hypertension, even in states where circulating levels of angiotensin II are suppressed. Dr. Taylor will use transgenic "knock-in" technology to define the functional significance of locally produced angiotensin II in the pathogenesis of atherosclerosis. This work will be important because it will provide information about how the events to be studied in projects 1 and 2 are initiated in pathophysiological states in vivo. These projects will be supported by two Cores that will provide expertise with detection of reactive oxygen species and histological analyses of relevant tissues. Overall, these studies will promote our understanding of the fundamental molecular mechanisms responsible for oxidant stress in the pathogenesis of vascular disease.